JP5802653B2 - Optical analysis apparatus, optical analysis method, and computer program for optical analysis - Google Patents

Description

  The present invention analyzes or analyzes the state of particles in a solution by detecting light from atoms, molecules or aggregates thereof (hereinafter referred to as “particles”) dispersed or dissolved in the solution. In particular, the present invention relates to an optical analysis apparatus, an optical analysis method, and a computer program therefor, and more particularly, using an optical system capable of detecting light from a minute region in a solution, such as an optical system of a confocal microscope or a multiphoton microscope. Of various particles, for example, biomolecules such as proteins, peptides, nucleic acids, lipids, sugar chains, amino acids or aggregates thereof, particulate objects such as viruses and cells, or non-biological particles The present invention relates to an optical analysis apparatus, an optical analysis method, and an optical analysis computer program capable of acquiring information useful for analyzing or analyzing states (interactions, binding / dissociation states, etc.).

  With the recent development of optical measurement technology, detection of weak light at the level of one photon or fluorescent single molecule using an optical system of a confocal microscope and an ultrasensitive photodetection technology capable of photon counting (one-photon detection)・ Measurement is possible. Thus, various apparatuses or methods for detecting intermolecular interactions such as biomolecules or binding / dissociation reactions using such a weak light measurement technique have been proposed. For example, in Fluorescence Correlation Spectroscopy (FCS; see, for example, Patent Documents 1 and 2 and Non-Patent Documents 1-3), a sample is obtained using an optical system of a laser confocal microscope and a photon counting technique. Fluorescence intensity from fluorescent molecules or fluorescently labeled molecules (fluorescent molecules, etc.) entering and exiting a microscopic area in the solution (focused area where the laser beam of the microscope is focused-called confocal volume) The average residence time (translational diffusion time) of the fluorescent molecules and the like in the minute region determined from the value of the autocorrelation function of the measured fluorescence intensity, and the average number of staying molecules Based on this, acquisition of information such as the speed, size, and concentration of movement of fluorescent molecules, etc., changes in the structure or size of molecules, molecular binding / dissociation reactions, dispersion / aggregation, etc. Detection is achieved of the people of the phenomenon. Fluorescence-Intensity Distribution Analysis (FIDA, for example, Patent Document 3) and Photon Counting Histogram (PCH, for example, Patent Document 4) are measured in the same manner as FCS. A histogram of the fluorescence intensity of the fluorescent molecules entering and exiting the volume is generated, and by fitting a statistical model formula to the distribution of the histogram, the average value of the intrinsic brightness of the fluorescent molecules and the confocal An average value of the number of molecules staying in the volume is calculated, and based on such information, a change in the structure or size of the molecule, a bound / dissociated state, a dispersed / aggregated state, and the like are estimated. Further, Patent Documents 5 and 6 propose a method of detecting a fluorescent substance based on the time lapse of a fluorescence signal of a sample solution measured using an optical system of a confocal microscope. Patent Document 7 describes the presence of fluorescent fine particles in a flow or on a substrate by measuring weak light from fluorescent fine particles circulated in a flow cytometer or fluorescent fine particles fixed on a substrate using a photon counting technique. A signal arithmetic processing technique for detecting the signal is proposed.

  In particular, according to a method using a micro area fluorescence measurement technique using an optical system of a confocal microscope such as FCS or FIDA and a photon counting technique, the sample required for measurement has an extremely low concentration compared with the conventional method. It can be very small (the amount used in one measurement is about several tens of μL), and the measurement time is greatly shortened (measurement of time on the order of seconds is repeated several times in one measurement). . Therefore, these technologies are particularly useful for analyzing rare or expensive samples often used in the field of medical and biological research and development, for clinical diagnosis of diseases, screening for physiologically active substances, etc. When the number is large, it is expected to be a powerful tool capable of performing experiments or inspections at a lower cost or faster than conventional biochemical methods.

JP-A-2005-09876 JP2008-292371 Patent No. 4023523 International Publication 2008-080417 JP2007-20565 JP2008-116440 JP-A-4-337446

Masataka Kaneshiro, Protein Nucleic Acid Enzyme Vol.44, No.9, pp.1431-1438 1999 FJMeyer-Alms, Fluorescence Correlation Spectroscopy, R. Rigler, Springer, Berlin, 2000, 204 -Page 224 Norio Kato, 4 people, Gene Medicine, Vol.6, No.2, pp.271-277

  In general, in the optical analysis techniques such as FCS, FIDA, and PCH described above, the magnitude of the temporal fluctuation of the measured fluorescence intensity is calculated by statistical processing, and the sample solution is based on the magnitude of the fluctuation. Various characteristics such as fluorescent molecules entering and exiting the minute region are determined. Therefore, in order to obtain a significant result in the above optical analysis technique, the concentration or number density of the fluorescent molecules to be observed in the sample solution must be as long as one second in the equilibrium state. In order for the number of fluorescent molecules that can be statistically processed within the measurement time to enter and exit the micro area, it is preferable that there is always approximately one fluorescent molecule in the micro area. It is preferable that the volume of the confocal volume is typically about 1 fL, so that the concentration of the fluorescent molecule or the like is preferably about 1 nM or more. In other words, when the concentration or number density of the particles to be observed in the sample solution is significantly lower than the level capable of statistical processing (for example, significantly lower than 1 nM), the observation target is measured in a minute region. A state that rarely enters in time occurs, and in the measurement result of fluorescence intensity, a state where the observation object is not present in the minute region is included for a long period of time, and is significant. Therefore, the optical analysis technique based on the statistical fluctuation of the fluorescence intensity as described above cannot expect a significant or accurate analysis result.

  In the method for detecting a fluorescent substance using the optical system of a confocal microscope described in Patent Documents 5 and 6, the statistical processing relating to the fluctuation of the fluorescence intensity as described above is not performed, and the measurement time over several seconds is used. The presence or absence of fluorescent molecules to be observed in the sample can be specified by the presence or absence of generation of a fluorescent signal with a significant intensity. The frequency of the fluorescent signal with a significant intensity and the number of fluorescent molecules or the like in the sample It is disclosed that a correlation can be obtained. In particular, Patent Document 6 suggests that detection sensitivity is improved by generating a random flow that stirs the sample solution. However, these methods are also limited to detecting the presence of fluorescent molecules or the like that enter the microregion stochastically by diffusion or random flow. The behavior cannot be grasped, and for example, particle counting and particle concentration or number density are not quantitatively calculated. Moreover, the technique described in Patent Document 7 is to individually detect the presence of fluorescent fine particles in a flow in a flow cytometer or fluorescent fine particles fixed on a substrate, and in a normal state in a sample solution. This is not a technique for detecting molecules or colloid particles dissolved or dispersed in the sample solution, that is, particles moving randomly in the sample solution. Quantitative calculation of concentration or number density has not been achieved. In addition, since the technique of Patent Document 7 includes a process such as measurement in a flow cytometer or immobilization of fluorescent particles on a substrate, the amount of sample required for inspection is optical analysis such as FCS, FIDA, PCH, etc. It is considered to require a complicated and sophisticated operation technique for the practitioner as well as the number of techniques.

  Thus, one object of the present invention does not include statistical processing, such as that performed in optical analysis techniques such as FCS, FIDA, PCH, etc., and therefore the concentration or number density of the particles to be observed is their optical analysis. It is to provide a novel optical analysis technique capable of detecting the state or characteristics of particles to be observed in a sample solution lower than the level handled by the technique.

  Another subject of the present invention is an optical analysis apparatus, method or computer program for realizing the above-described novel optical analysis technology, which is measured in the same manner as optical analysis technologies such as FCS, FIDA, and PCH. The amount of sample required for the measurement may be a very small amount (for example, about several tens of μL), and the measurement time is short and the optical analysis capable of quantitatively specifying the characteristics such as the concentration or number density of the particles to be observed An apparatus, an optical analysis method, or a computer program for optical analysis is provided.

  Generally speaking, in the present invention, an optical system capable of detecting light from a minute region in a solution, such as an optical system of a confocal microscope or a multiphoton microscope, is used to randomly disperse in a sample solution. This is an optical analysis technique for detecting light from particles that emit moving light (hereinafter referred to as “luminescent particles”), while moving the position of a minute region that is a light detection region in a sample solution. That is, light from the micro area is detected while scanning the sample solution by the micro area, and thereby, the luminescent particles crossing the micro area are individually detected to count the luminescent particles and emit light in the sample solution. A new type of optical analysis technique is proposed that enables the acquisition of information on the concentration or number density of particles.

  According to the present invention, as one embodiment, an optical analyzer that detects light from luminescent particles that are dispersed in a sample solution and move randomly using an optical system of a confocal microscope or a multiphoton microscope, A light detection region moving unit that moves the position of the light detection region of the optical system in the sample solution, a light detection unit that detects light from the light detection region, and a light detection region in the sample solution. Generate time-series light intensity data of light from the light detection area detected by the light detection unit while moving the position, and individually generate light signals from each of the luminescent particles in the time-series light intensity data A signal processing unit for detecting, and the signal processing unit calculates a light intensity characteristic value indicating the presence or absence of light from a single light emitting particle for each time interval of a predetermined width in time-series light intensity data. Using these characteristic values, the optical signal from each of the luminescent particles can be individually And wherein that output is provided. In such a configuration, “the luminous particles dispersed in the sample solution and moving randomly” are particles that emit light, such as atoms, molecules or aggregates thereof dispersed or dissolved in the sample solution, Any particle may be used as long as it is not fixed to the substrate or the like and freely moves in the solution in Brownian motion. Such luminescent particles are typically fluorescent particles, but may be particles that emit light by phosphorescence, chemiluminescence, bioluminescence, light scattering, or the like. The “light detection area” of the optical system of a confocal microscope or multiphoton microscope is a minute area in which light is detected in those microscopes. When illumination light is given from an objective lens, the illumination light is Corresponds to the focused region (in the confocal microscope, it is determined in particular by the positional relationship between the objective lens and the pinhole. When the luminescent particles emit light without illumination light, for example, chemiluminescence or biological In the case of particles that emit light by light emission, illumination light is not required in the microscope.) In this specification, “optical signal” means “signal representing light”.

  As understood from the above, in the basic configuration of the apparatus of the present invention, first, the position of the light detection region is moved in the sample solution, that is, the sample solution is scanned by the light detection region. However, light is detected sequentially. Then, when the moving light detection region includes light emitting particles moving at random, light from the light emitting particles is detected by the light detection unit. The presence can be detected. In this regard, more specifically, in a time interval in which light from a single luminescent particle arrives at the light detection unit in the signal output in time series from the light detection unit, that is, light intensity data. In this case, the value of the output signal of the light detection unit changes in a pulse shape, and has a characteristic that is different from the time interval in which light from the luminescent particles in which only noise occurs does not arrive. Therefore, the signal processing unit of the apparatus sequentially generates time-series light intensity data from the signals detected by the light detection unit, and in the time-series light intensity data, each time-series light intensity data is A characteristic value of light intensity representing the presence / absence of light from one luminescent particle is calculated, and the optical signal from each of the luminescent particles is individually detected using the characteristic value.

  As such a characteristic value, the size in a time interval of a predetermined width where light from a single light emitting particle exists is a time interval of a predetermined width where light from a single light emitting particle does not exist Any value that is larger than the size in can be used. In this case, when the characteristic value of the light intensity is larger than a predetermined threshold, it is determined that the light signal from the luminescent particles exists in a time interval of a predetermined width. Such characteristic values include, for example, an integrated value of light intensity, a median value of light intensity, an average value of light intensity, a standard deviation of light intensity, a dispersion of light intensity, and an entropy of light intensity. Or the number of particles calculated from the value when the correlation time of the maximum value of the light intensity and the autocorrelation function of the light intensity is zero. Typically, the photodetection unit detects light from the photodetection region by photon counting. Therefore, in this case, time-series light intensity data becomes time-series photon count data. Therefore, the characteristic value of the light intensity includes the total value of the photons in the time interval of the predetermined width, the median value of the photons, the average value of the photons, the standard deviation of the photons, the dispersion of the photons, and the entropy of the photons. Further, it may be a value selected from the group of particle numbers calculated from the autocorrelation function value of the photon number with the maximum value of the photon number and the correlation time being zero.

  Further, in the case of the method for detecting the signal of the luminescent particle using the characteristic value for each time interval of the predetermined width as described above, the time required for the predetermined width from the time when the single light emitting particle enters the light detection region to the time when it escapes. If it is shorter than the width, the light of one luminescent particle will be detected over two or more time intervals, and if the predetermined width is long enough to include the arrival times of the light of a plurality of luminescent particles In one case, information on a plurality of luminescent particles is included, and in any case, a relationship in which one luminescent particle corresponds to one time interval is not established. The signal processing for individually detecting the optical signal from each becomes complicated. Therefore, the predetermined width of the time interval for calculating the characteristic value is preferably set to be substantially longer than the time width required from the time when one luminescent particle enters the light detection region to the time when it escapes. In addition, it is set to be substantially shorter than the time width from the time when one light emitting particle enters the light detection region to the time when another light emitting particle enters the light detection region. In other words, the predetermined width of the time interval is set such that the lower limit is substantially longer than the time width of the optical signal of one luminescent particle, and the upper limit is the number of luminescent particles included in each time interval. Is set to be substantially 1 or less. In the above expression, “it is set to be substantially longer than the time width required from the time when one light emitting particle enters the light detection region to the time when it exits” or “the light signal of one light emitting particle”. `` Set to be substantially longer than the time width of '' means that the predetermined width is set longer than the time width of the optical signal of most luminescent particles, that is, a time width longer than the predetermined width within an error range. It means that the presence of an optical signal of the luminescent particles having Also, “substantially set to be shorter than the time width from the time when one luminescent particle enters the light detection region to the time when another luminescent particle enters the light detection region” or “included in each time interval “Set so that the number of luminescent particles to be substantially reduced to 1 or less” means to set the number of luminescent particles found in most time intervals to be 1 or less, that is, within an error range. This means that a time interval including two or more luminescent particles is allowed. According to this configuration, when one time interval in which light from the luminescent particles exists is determined, in most cases, the optical signal included in the time interval is an optical signal from one luminescent particle. As a result, the presence of one luminescent particle is confirmed, which facilitates the process of individually detecting the luminescent particle, which is advantageous.

  As described above, when the luminescent particles moving randomly in the sample solution can be individually detected, various information on the state of the luminescent particles in the solution is acquired. Specifically, for example, in the apparatus of the present invention described above, the signal processing unit counts the number of light signals from the individually detected luminescent particles and is detected while the position of the light detection region is moving. The number of luminescent particles may be counted (counting of luminescent particles). In that case, as described above, when the light emitting particles are individually detected using the characteristic value of the light intensity for each time interval of the predetermined width, the number of time intervals of the predetermined width having the light signal from the light emitting particles is the light emission. Since this corresponds to the number of particles, the number of time intervals having a predetermined width having the optical signal from the luminescent particles is counted, and the number of luminescent particles detected during the movement of the position of the light detection region is counted. According to such a configuration, information on the number density or concentration of the luminescent particles in the sample solution can be obtained by combining the number of luminescent particles and the amount of movement of the position of the light detection region. In particular, if the total volume of the movement locus of the light detection region is specified by any method, for example, by moving the position of the light detection region at a predetermined speed, the number density or concentration of the luminescent particles is reduced. It can be calculated specifically. Of course, the absolute number density value or concentration value is not directly determined, but the relative number density or concentration ratio with respect to a plurality of sample solutions or a standard sample solution serving as a reference for the concentration or number density is calculated. It may be like this.

  In the apparatus of the present invention, the moving speed of the position of the light detection region in the sample solution can be appropriately changed based on the characteristics of the luminescent particles or the number density or concentration in the sample solution. Good. As will be appreciated by those skilled in the art, the manner of light detected from the luminescent particles can vary depending on the properties of the luminescent particles or the number density or concentration in the sample solution. In particular, as the moving speed of the light detection region increases, the amount of light obtained from one light emitting particle decreases, so that the light from one light emitting particle can be measured with high accuracy or sensitivity. It is preferable that the moving speed can be appropriately changed.

  Further, in the apparatus of the present invention, the moving speed of the position of the light detection region in the sample solution is preferably the diffusion moving speed of the luminescent particles to be detected (average moving speed of particles due to Brownian motion). Higher than. As described above, the apparatus of the present invention detects the light emitted from the light emitting particles when the light detection region passes the position where the light emitting particles exist, and individually detects the light emitting particles. However, if the luminescent particles move randomly in the solution due to Brownian motion and enter and exit the photodetection region multiple times, an optical signal (representing the presence of the luminescent particles) from one luminescent particle multiple times Is detected, and it becomes difficult to make the detected optical signal correspond to the presence of one luminescent particle. Therefore, as described above, the moving speed of the light detection region is set to be higher than the diffusion moving speed of the light emitting particles, and thereby one light emitting particle can correspond to one light signal (representing the presence of the light emitting particle). It becomes possible. Since the diffusion movement speed varies depending on the luminescent particles, as described above, according to the characteristics of the luminescent particles (particularly, the diffusion constant), the apparatus of the present invention can appropriately change the movement speed of the light detection region. It is preferable to be configured.

  The movement of the position of the light detection region may be performed by an arbitrary method. For example, the position of the light detection region may be changed by changing the optical path using a galvanometer mirror employed in a laser scanning optical microscope, or the light detection region may be fixed, The position of the sample solution may be moved (for example, by moving a microscope stage) to move the position of the light detection region in the sample solution. The movement trajectory of the position of the light detection region may be arbitrarily set, and may be selected from, for example, a circle, an ellipse, a rectangle, a straight line, and a curve.

  In the above-described apparatus of the present invention, the light detection is performed while moving the position of the light detection region in the sample solution, and the light signal from each light emitting particle is individually detected. The processing can also be realized by a general-purpose computer. Therefore, according to another aspect of the present invention, for optical analysis for detecting light from luminous particles dispersed and moving randomly in a sample solution using an optical system of a confocal microscope or a multiphoton microscope. A computer program for moving the position of the light detection area of the optical system in the sample solution and light from the light detection area during movement of the position of the light detection area in the sample solution. A procedure for detecting and generating time-series light intensity data, and calculating a characteristic value of light intensity that indicates the presence or absence of light from a single light emitting particle for each time interval of a predetermined width in the time-series light intensity data And a computer program for causing a computer to execute a procedure for individually detecting a light signal from each light emitting particle based on a characteristic value of light intensity for each time interval of a predetermined width. .

  In such a computer program, the number of light signals from the light-emitting particles detected individually or the number of time intervals having a predetermined width having the light signals from the light-emitting particles is counted to determine the position of the light detection region. A procedure for counting the number of luminescent particles detected during movement of the sample and / or a procedure for determining the number density or concentration of luminescent particles in the sample solution based on the number of detected luminescent particles. . Typically, in the procedure of detecting light from the light detection region and generating time-series light intensity data, light from the light detection region is detected by photon counting, in which case the time-series light intensity is detected. The data is time-series photon count data. In the procedure of moving the position of the light detection region, the position of the light detection region may be moved at a predetermined speed or at a speed faster than the diffusion movement speed of the luminescent particles. The moving speed of the position of the detection region may be set based on the characteristics of the luminescent particles or the number density or concentration in the sample solution. The movement locus of the position of the light detection region may be selectable from a circle, an ellipse, a rectangle, a straight line, and a curve.

  Further, even in the above computer program, the characteristic value of the light intensity in the time interval of the predetermined width is obtained from the light emitted from a single light emitting particle when light from the single light emitting particle exists. It may be a value that is greater than when no light is present, and when the light intensity characteristic value is greater than a predetermined threshold, it may be determined that an optical signal from the luminescent particles is present in a time interval of a predetermined width. Specifically, such characteristic values include the integrated value of light intensity, the median value of light intensity, the average value of light intensity, the standard deviation of light intensity, the dispersion of light intensity, the light intensity, Any of the number of particles calculated from the entropy of the intensity, the maximum value of the light intensity, and the autocorrelation function value of the light intensity with the correlation time being 0 may be adopted. In particular, when the time-series light intensity data is time-series photon count data, the characteristic value of the light intensity includes the total number of photons, the median of the number of photons, and the average of the number of photons in a predetermined time interval. Selected from the group of particle numbers calculated from the value, the standard deviation of the number of photons, the dispersion of the number of photons, the entropy of the number of photons, the maximum value of the number of photons and the autocorrelation function value of the number of photons with zero correlation time. It may be a value. Further, the predetermined width of the time interval is preferably set to be substantially longer than a time width required from the time when one luminescent particle enters the light detection region to the time when it exits, and It is set to be substantially shorter than the time width from the time when one luminescent particle enters the light detection region to the time when another luminescent particle enters the light detection region.

  Furthermore, according to the above-described apparatus or computer program of the present invention, as described above, the light detection is performed while moving the position of the light detection region in the sample solution, and the light signal from each light emitting particle is individually received. A novel optical analysis method for detection is realized. Therefore, the optical analysis method for detecting light from the luminous particles dispersed and moving randomly in the sample solution using the optical system of the confocal microscope or multiphoton microscope of the present invention is the above-described optical analysis method in the sample solution. The process of moving the position of the light detection area of the optical system, and the process of generating light in time series by detecting the light from the light detection area while moving the position of the light detection area in the sample solution In the time-series light intensity data, the process of calculating the characteristic value of the light intensity indicating the presence or absence of light from a single light emitting particle for each time interval of a predetermined width, and the light intensity of each time interval of a predetermined width And a step of individually detecting optical signals from the individual luminescent particles based on the characteristic values.

  Even in such a method, the number of light signals from individually detected luminescent particles is counted, or the number of time intervals having a predetermined width having light signals from luminescent particles is counted to determine the position of the light detection region. A step of counting the number of luminescent particles detected during movement and / or a step of determining the number density or concentration of luminescent particles in the sample solution based on the number of detected luminescent particles may be included. Typically, in the process of detecting light from the light detection region and generating time series light intensity data, the light from the light detection region is detected by photon counting, in which case the time series light intensity is detected. The data is time-series photon count data. Further, in the process of moving the position of the light detection region, the position of the light detection region may be moved at a predetermined speed or at a speed faster than the diffusion movement speed of the luminescent particles. The moving speed of the position of the detection region may be set based on the characteristics of the luminescent particles or the number density or concentration in the sample solution. The movement locus of the position of the light detection region may be selectable from a circle, an ellipse, a rectangle, a straight line, and a curve.

  Further, even in the above method, the characteristic value of the light intensity in the time interval of the predetermined width is that the light from a single luminescent particle is present when light from a single luminescent particle is present. It may be a value that is larger than the case where no light exists, and when the light intensity characteristic value is larger than a predetermined threshold value, it may be determined that the light signal from the luminescent particles is present in a time interval of a predetermined width. Specifically, such characteristic values include the integrated value of light intensity, the median value of light intensity, the average value of light intensity, the standard deviation of light intensity, the dispersion of light intensity, the light intensity, Any of the number of particles calculated from the entropy of the intensity, the maximum value of the light intensity, and the autocorrelation function value of the light intensity with the correlation time being 0 may be adopted. In particular, when the time-series light intensity data is time-series photon count data, the characteristic value of the light intensity includes the total number of photons, the median of the number of photons, and the average of the number of photons in a predetermined time interval. Selected from the group of particle numbers calculated from the value, the standard deviation of the number of photons, the dispersion of the number of photons, the entropy of the number of photons, the maximum value of the number of photons and the autocorrelation function value of the number of photons with zero correlation time. It may be a value. Further, the predetermined width of the time interval is preferably set to be substantially longer than a time width required from the time when one luminescent particle enters the light detection region to the time when it exits, and It is set to be substantially shorter than the time width from the time when one light emitting particle enters the light detection region to the time when another light emitting particle enters the light detection region.

  The optical analysis technique realized by the above-described apparatus, method or computer program according to the present invention is the same as in the case of optical analysis techniques such as FCS, FIDA, PCH, etc. Since it is configured to detect light from the light detection region of the microscope, the amount of the sample solution may be small as well. However, in the present invention, since statistical processing such as calculating fluctuations in fluorescence intensity is not performed, the photoanalysis technique of the present invention uses photoanalysis such as the number density or concentration of luminescent particles of FCS, FIDA, PCH, etc. Applicable to sample solutions significantly below the level required for the technology.

  Further, in the present invention, each of the luminous particles dispersed or dissolved in the solution is individually detected. Therefore, the information is used to quantitatively count the luminous particles or the luminous particles in the sample solution. It is possible to calculate the concentration or the number density of the above or to acquire information on the concentration or the number density. For example, in the techniques described in Patent Documents 5 and 6, the correlation between the total value of the frequency of fluorescent signals having an intensity equal to or higher than a predetermined threshold within a predetermined time and the number of particles such as fluorescent molecules in the sample solution is obtained. However, it is not possible to grasp the dynamic behavior of particles passing through the measurement region (whether the particles have passed straight through the measurement region or whether the particles have stayed in the measurement region). Since the correspondence between the fluorescence signal having an intensity equal to or higher than a predetermined threshold and the particles passing through the measurement region is unclear, counting of luminescent particles is impossible in principle, and the concentration of particles in the sample solution is accurate. It was difficult to decide well. However, according to the present invention, since the luminescent particles that pass through the light detection region and the arrival time of the optical signal to the light detection unit are associated with each other one by one, the luminescent particles are detected one by one. It is possible to count luminous particles that are dispersed and move in a random manner, and it is possible to determine the concentration or number density of particles in a sample solution with higher accuracy than before.

  The optical analysis technique of the present invention typically involves the production of particulate biological objects such as proteins, peptides, nucleic acids, lipids, sugar chains, amino acids or aggregates thereof, viruses, cells and the like. Used for analysis or analysis of conditions in solution, but may be used for analysis or analysis of conditions in solution of non-biological particles (eg, atoms, molecules, micelles, metal colloids, etc.) It should be understood that such a case also belongs to the scope of the present invention.

  Other objects and advantages of the present invention will become apparent from the following description of preferred embodiments of the present invention.

FIG. 1A is a schematic diagram of the internal structure of an optical analyzer according to the present invention. FIG. 1B is a schematic diagram of a confocal volume (observation region of a confocal microscope). FIG. 1C is a schematic diagram of a mechanism for changing the direction of the mirror 7 to move the position of the light detection region in the sample solution. FIG. 1D is a schematic diagram of a mechanism for moving the position of the photodetection region in the sample solution by moving the horizontal position of the microplate. FIGS. 2A and 2B are a schematic diagram for explaining the principle of light detection by the optical analysis technique according to the present invention and a schematic diagram of a temporal change in measured light intensity, respectively. FIG. 2C is a diagram for explaining the principle of individually detecting light from the luminescent particles from the light intensity data. FIG. 2 (D) shows the predetermined width of the time interval and the probability that the light of two or more luminescent particles has entered at the same time (the ratio that a plurality of luminescent particles are included in the time interval of one predetermined width ω). Showing the relationship. FIGS. 3A and 3B are a model diagram in the case where the luminescent particle crosses the light detection region while performing a Brownian motion, and a diagram showing an example of a temporal change in photon count (light intensity) in that case. is there. FIGS. 4A and 4B show models in which the luminescent particles cross the light detection region by moving the position of the light detection region in the sample solution at a speed faster than the diffusion movement speed of the luminescent particles. It is a figure which shows the example of the time change of a figure and the photon count (light intensity) in that case. FIG. 5A is a diagram showing the entire time-series photon count data measured by the optical analysis technique of the present invention, and FIG. 5B is an enlarged view of a part of the time-series photon count data. It is a figure and shows the various characteristic values calculated about the section currently referred. FIG. 6 shows the number of luminescent particles individually detected in the time-series photon count data using the characteristic values calculated according to the present invention for each solution having a luminescent particle concentration of 10 fM to 100 pM. FIG. 7 is a diagram showing the relationship between the predetermined width of the time interval and the number of intervals in which the presence of luminescent particles is detected in the time-series photon count data obtained by the optical analysis technique according to the present invention. FIG. 8 is an example of the time change of the photon count (light intensity) obtained in the conventional optical analysis technique for calculating the fluctuation of the fluorescence intensity. FIG. 8A shows that the concentration of particles in the sample is sufficient. This is a case where measurement accuracy is given, and (B) is a case where the concentration of particles in the sample is significantly lower than in (A).

1 ... Optical analyzer (confocal microscope)
DESCRIPTION OF SYMBOLS 2 ... Light source 3 ... Single mode optical fiber 4 ... Collimator lens 5 ... Dichroic mirror 6, 7, 11 ... Reflection mirror 8 ... Objective lens 9 ... Microplate 10 ... Well (sample solution container)
DESCRIPTION OF SYMBOLS 12 ... Condenser lens 13 ... Pinhole 14 ... Barrier filter 15 ... Multi-mode optical fiber 16 ... Photo detector 17 ... Mirror deflector 17a ... Stage position change device 18 ... Computer

  Hereinafter, preferred embodiments of the present invention will be described in detail.

Configuration of Optical Analysis Apparatus The optical analysis apparatus that realizes the optical analysis technique according to the present invention is capable of executing FCS, FIDA, and the like as schematically illustrated in FIG. The apparatus may be a combination of an optical system of a confocal microscope and a photodetector. Referring to FIG. 1, optical analysis apparatus 1 includes optical systems 2 to 17 and a computer 18 for controlling the operation of each part of the optical system and acquiring and analyzing data. The optical system of the optical analyzer 1 may be the same as the optical system of a normal confocal microscope, in which the laser light (Ex) emitted from the light source 2 and propagated through the single mode fiber 3 is a fiber. The light is emitted as a divergent light at an angle determined by a specific NA at the outgoing end of the light, becomes parallel light by the collimator 4, is reflected by the dichroic mirror 5, the reflection mirrors 6, 7, and the objective lens 8. Is incident on. Above the objective lens 8, a microplate 9 in which sample containers or wells 10 into which a sample solution of 1 to several tens μL is dispensed is typically arranged is emitted from the objective lens 8. The laser light is focused in the sample solution in the sample container or well 10 to form a region with high light intensity (excitation region). In the sample solution, light-emitting particles that are the observation target, typically particles to which fluorescent labels such as fluorescent particles or fluorescent dyes are added are dispersed or dissolved, and these light-emitting particles enter the excitation region. In the meantime, the luminescent particles are excited and light is emitted. The emitted light (Em) passes through the objective lens 8 and the dichroic mirror 5, is reflected by the mirror 11, is collected by the condenser lens 12, passes through the pinhole 13, and passes through the barrier filter 14. (Here, only the light component of a specific wavelength band is selected.), Introduced into the multimode fiber 15, reaches the photodetector 16, is converted into a time-series electrical signal, and then to the computer 18. Input and processing for optical analysis is performed in a manner described later. As is known to those skilled in the art, in the above configuration, the pinhole 13 is disposed at a position conjugate with the focal position of the objective lens 8, and as a result, as shown in FIG. Only the light emitted from the focal region of the laser beam, that is, the excitation region as schematically shown, passes through the pinhole 13, and the light from other than the excitation region is blocked. The focal region of the laser light illustrated in FIG. 1B is usually a light detection region in the present optical analyzer having an effective volume of about 1 to 10 fL (typically, the light intensity is in the region). It has a Gaussian distribution with the center at the apex.The effective volume is the volume of a substantially elliptical sphere bounded by the plane where the light intensity is 1 / e ² )), and is called confocal volume. Further, in the present invention, light from one luminescent particle, for example, faint light from one fluorescent dye molecule is detected. Therefore, the photodetector 16 is preferably an ultra-high light that can be used for photon counting. A sensitive photodetector is used. When the light detection is based on photon counting, the light intensity is measured in a manner in which the number of photons arriving at the photodetector is sequentially measured for a predetermined unit time (BIN TIME) over a predetermined time. Executed. Therefore, in this case, the time-series light intensity data is time-series photon count data.

  Further, in the optical system of the above optical analyzer, a mechanism for scanning the sample solution with the light detection region, that is, for moving the position of the focal region, that is, the light detection region in the sample solution. Is provided. As a mechanism for moving the position of the light detection region, for example, a mirror deflector 17 that changes the direction of the reflection mirror 7 may be employed as schematically illustrated in FIG. A method of moving the absolute position of the light detection area). Such a mirror deflector 17 may be the same as a galvanometer mirror device provided in a normal laser scanning microscope. Alternatively, as another embodiment, as illustrated in FIG. 1D, the horizontal position of the container 10 (microplate 9) into which the sample solution is injected is moved to detect light in the sample solution. A stage position changing device 17a that moves the relative position of the region may be employed (a method of moving the absolute position of the sample solution). In any case, the mirror deflector 17 or the stage position changing device 17a cooperates with the light detection by the light detector 16 under the control of the computer 18 in order to achieve a desired movement pattern of the position of the light detection region. Driven. The movement trajectory of the position of the light detection region may be arbitrarily selected from a circle, an ellipse, a rectangle, a straight line, a curve, or a combination thereof (so that various movement patterns can be selected by the program in the computer 18). It may be.) Although not shown, the position of the light detection region may be moved in the vertical direction by moving the objective lens 8 or the stage up and down.

  In the case where the luminescent particles that are the observation object emit light by multiphoton absorption, the above optical system is used as a multiphoton microscope. In that case, since there is light emission only in the focal region (light detection region) of the excitation light, the pinhole 13 may be removed. In addition, when the luminescent particles to be observed emit light regardless of excitation light due to chemiluminescence or bioluminescence phenomenon, the optical systems 2 to 5 for generating excitation light may be omitted. When the luminescent particles emit light by phosphorescence or scattering, the optical system of the confocal microscope is used as it is. Furthermore, in the optical analyzer 1, as shown in the figure, a plurality of excitation light sources 2 may be provided, and the wavelength of the excitation light may be appropriately selected according to the excitation wavelength of the luminescent particles. Similarly, a plurality of photodetectors 16 may be provided, and when a plurality of types of light emitting particles having different wavelengths are included in the sample, light can be separately detected based on the wavelengths. It's okay.

Principle of optical analysis technology of the present invention The spectroscopic analysis technology such as FCS, FIDA and the like is superior to the conventional biochemical analysis technology in that the required amount of sample is extremely small and inspection can be performed quickly. ing. However, in the spectroscopic analysis techniques such as FCS and FIDA, in principle, the concentration and characteristics of the observation target particles are calculated based on the fluctuation of the fluorescence intensity. Therefore, in order to obtain an accurate measurement result, the sample solution As schematically illustrated in FIG. 8A, the concentration or number density of the observation target particles therein is always about one observation target particle in the light detection region CV during the measurement of the fluorescence intensity. As shown on the right side of the figure, it is required that a significant light intensity (photon count) is always detected during the measurement time. If the concentration or number density of the observation target particles is lower than that, for example, as shown in FIG. 8B, the observation target particles are at a level that only occasionally enters the light detection region CV. As illustrated on the right side of the figure, a significant light intensity (photon count) appears only in a part of the measurement time, making it difficult to accurately calculate the fluctuation of the light intensity. In addition, if the concentration of the observation target particle is much lower than the level at which about one observation target particle is always present in the light detection area during measurement, the influence of the background is considered in the calculation of the light intensity fluctuation. The measurement time is long in order to obtain significant light intensity data sufficient for calculation.

  Therefore, in the present invention, even when the concentration of the observation target particles is lower than the level required by the spectral analysis technology such as FCS or FIDA as described above, the characteristics such as the number density or the concentration of the observation target particles. An optical analysis technique based on a novel principle that enables detection of the light is proposed.

  In the optical analysis technique of the present invention, as a process to be executed, in short, a mechanism (mirror deflector 17 or stage position changing device 17a) for moving the position of the light detection region is driven, As schematically illustrated in FIG. 2, light detection is performed while moving the position of the light detection region CV in the sample solution, that is, while scanning the sample solution by the light detection region CV. Is done. Then, for example, as shown in FIG. 2A, while the light detection region CV moves (time to t2 in the drawing), it passes through the region where one luminescent particle (fluorescent dye in the drawing) exists. At this time (t1), as shown in FIG. 2B, a significant light intensity (Em) is detected as a pulse signal. Thus, the movement of the position of the light detection region CV and the light detection are executed, and the significant light intensity, that is, the pulse-like signal appearing in the meantime as illustrated in FIG. 2B is detected one by one. Thus, the luminescent particles are individually detected, and by counting the number thereof, information on the number of luminescent particles existing in the measured region, or the concentration or number density can be acquired. In the principle of the optical analysis technique of the present invention, statistical calculation processing such as calculation of fluctuations in fluorescence intensity is not performed, and luminescent particles are detected one by one, and FCS, FIDA, etc. are sufficient. It should be understood that information on the concentration or number density of luminescent particles can be obtained even in a sample solution having a low concentration of luminescent particles (observation target particles) that cannot be analyzed with high accuracy.

  In the actual light intensity data (photon count data), as illustrated in FIG. 2C, in addition to the light from the luminescent particles, noise (thermal noise of the photodetector, background light) Therefore, it is necessary to distinguish the optical signal from the luminescent particles from the noise and detect the presence of the optical signal from the luminescent particles. Therefore, as one of the methods for extracting the presence of the optical signal from such luminescent particles, in the present embodiment, in the time-series light intensity data, it is sequentially applied for each time interval of a predetermined width. In addition, a light intensity characteristic value indicating the presence or absence of light from a single luminescent particle is calculated using the light intensity value (number of photons) in the time interval. By referring to the magnitude of the characteristic value of the light intensity, it is possible to detect whether or not there is an optical signal from a single light emitting particle for each time interval of a predetermined width. Detected one by one. The characteristic value of the light intensity that indicates the presence or absence of light from a single luminescent particle is typically the presence of a light signal from a single luminescent particle and the presence of a light signal from a single luminescent particle. It may be an arbitrary value that increases as compared to the case where only noise is present (for example, when only noise is present). For example, the integrated value of the light intensity and the median value of the light intensity in a predetermined time interval Average value of light intensity, standard deviation value of light intensity (SD value), dispersion value of light intensity, entropy of light intensity (EP value), maximum value of light intensity and autocorrelation of light intensity with correlation time being zero It may be the number of particles calculated from the function value. In particular, in the present embodiment, since the light intensity data is photon count data by photon counting, the characteristic values are a total value of the number of photons in a time interval of a predetermined width, a median value of the number of photons, an average value of the number of photons, Select from the group of particle numbers calculated from the standard deviation value of the photon number, the dispersion value of the photon number, the entropy value of the photon number, the maximum value of the photon number, and the autocorrelation function value of the photon number with zero correlation time The value may be

  In addition, the predetermined width of the time interval for calculating the characteristic value is preferably substantially longer than the time width required from the time when one luminescent particle enters the light detection region to the time when it exits, And it is set substantially shorter than the time width from the time of the approach of one luminescent particle to the photodetection region until the time of the entrance of another luminescent particle to the photodetection region. When the predetermined width is set in this way, one time interval in which the light signal of the luminescent particles exists corresponds to the presence of one luminescent particle, that is, the time interval and the presence of the luminescent particles have a one-to-one relationship. This is advantageous because the processing in the counting of the light emitting particles is simplified. (If the predetermined width of the time interval is shorter than the time width required from the time when one light emitting particle enters the light detection region to the time when it exits, the light signal of one light emitting particle is spread over a plurality of time intervals. Therefore, a process for associating a plurality of time intervals with one luminescent particle is necessary, and if the predetermined width of the time interval is different from the time when one luminescent particle enters the light detection region, If it is longer than the time width until the light emitting particle enters the light detection region, the light signal of the plurality of light emitting particles exists in one time interval, and it is difficult to detect the light emitting particles one by one. Will be.)

The lower limit ωlow of the predetermined width is assumed when the luminescent particles pass linearly through the center of the light detection region (see (1) Measurement of the light intensity of the sample solution and FIG. 4 below), for example, the light detection region. Using the diameter 2 Wo and the moving speed u
ωlow = 2Wo / u (1)
May be set.

The upper limit ωhigh of the predetermined width is a predetermined width in which the number of luminescent particles included in each time interval is substantially 1 or less. The upper limit ωhigh of the predetermined width can be determined by any of the following methods.
(1) When it is assumed that the luminescent particles are uniformly dispersed in the sample solution, the number n of luminescent particles detected during the movement of the light detection region in the time interval of the predetermined width ω is:
n = cN _A Suω (2a)
Given by. Here, c is the molar concentration of the luminescent particles, N _A is Avogadro's number, S is a sectional area of the light detection region. Assuming that there is one luminescent particle (n = 1) in a time interval with a predetermined width ω, approximating S˜πWo ² , the predetermined width ω is
_{ω = 1 / (cN A πWo} 2 u) ... (2b)
Given by. Accordingly, the predetermined width ωhigh is set to a value ω calculated by the equation (2b) using the expected concentration c of the luminescent particles, or a value smaller than that in consideration of the variation in the location of the luminescent particles. May be set.

となるので、所定幅ωの時間区間に出現する発光粒子数が２以上である確率ｐ_＞１、即ち、複数の発光粒子が一つの所定幅ωの時間区間に包含される割合は、

となる。図２（Ｄ）は、所定幅ωに対する式（２ｄ）の変化の例を表した図である（ｃ＝１０ｐＭ、ｕ＝１５ｍｍ／ｓ、Ｗｏ＝１μｍとして算出した。）。同図を参照して、図示の例では、所定幅ω＝２００μ秒とすると、５％の時間区間が複数の発光粒子を包含する可能性があることが理解される。かくして、所定幅ωhighは、予想される発光粒子の濃度ｃを用いて、上記の式（２ｄ）に於いて、許容誤差を与える所定幅ωに設定されてよい。(2) When it is assumed that luminescent particles enter a time interval of a predetermined width in a Poisson distribution, the average number n of luminescent particles detected during the movement of the light detection region in the time interval of the predetermined width ω is (2a). On the other hand, the probability pk that the number of luminescent particles appearing in the time interval of the predetermined width ω is k is:

Therefore, the probability p _{> 1} that the number of luminescent particles appearing in the time interval of the predetermined width ω is 2 or more, that is, the ratio of the plurality of luminescent particles included in the time interval of one predetermined width ω is:

It becomes. FIG. 2D is a diagram showing an example of the change in the expression (2d) with respect to the predetermined width ω (calculated as c = 10 pM, u = 15 mm / s, Wo = 1 μm). Referring to the figure, in the illustrated example, it is understood that a time interval of 5% may include a plurality of luminescent particles when the predetermined width ω = 200 μsec. Thus, the predetermined width ωhigh may be set to the predetermined width ω that gives an allowable error in the above equation (2d) using the expected concentration c of the luminescent particles.

Operation and processing procedure of the optical analyzer of the present invention In the optical analysis according to the present invention using the optical analyzer 1 illustrated in FIG. 1 (A), specifically, (1) Luminescent particles (observation object) The light intensity measurement process of the sample solution containing particles) and (2) the measured light intensity analysis process are executed.

(1) Measurement of the light intensity of the sample solution The particles to be observed in the optical analysis technique of the present invention are particles such as dissolved molecules that are dispersed in the sample solution and move randomly in the solution. For example, a biomolecule such as a protein, peptide, nucleic acid, lipid, sugar chain, amino acid or aggregate thereof, virus, cell, metal colloid, or other non-biological molecule may be used. And so on. If the particle to be observed is not a particle that emits light, a luminescent label (fluorescent molecule, phosphorescent molecule, chemical / bioluminescent molecule) is added to the particle to be observed in any form. Used. The sample solution is typically an aqueous solution, but is not limited thereto, and may be an organic solvent or any other liquid.

  The light intensity in the optical analysis of the present invention is measured by driving the mirror deflector 17 or the stage position changing device 17a during the measurement to move the position of the light detection region in the sample solution (scanning in the sample solution). ) May be performed in the same manner as the light intensity measurement process in FCS or FIDA. In the operation process, typically, after injecting the sample solution into the well 10 of the microplate 9 and placing it on the stage of the microscope, the user inputs an instruction to start measurement to the computer 18. Then, the computer 18 stores a program stored in a storage device (not shown) (procedure for moving the position of the light detection region in the sample solution, and from the light detection region during movement of the position of the light detection region). In accordance with a procedure for detecting light and generating time-series light intensity data, irradiation of excitation light and measurement of light intensity in the light detection region in the sample solution are started. During the measurement, under the control of the processing operation according to the program of the computer 18, the mirror deflector 17 or the stage position changing device 17a drives the mirror 7 (galvanomirror) or the microplate 9 on the stage of the microscope, and the wells. 10, the position of the light detection region is moved, and at the same time, the light detector 16 sequentially converts the detected light into an electric signal and transmits it to the computer 18. In this manner, time-series light intensity data is generated from the transmitted optical signal and stored. Typically, the photodetector 16 is an ultra-sensitive photodetector that can detect the arrival of one photon, and therefore when the light detection is based on photon counting, the time-series light intensity data is a time-series data. It may be photon count data.

  The moving speed of the position of the light detection region during the measurement of the light intensity may optionally be a predetermined speed set, for example, experimentally or to suit the purpose of the analysis. When acquiring information on the number density or concentration based on the number of detected luminescent particles, the size or volume of the region through which the light detection region has passed is required, so that the movement distance can be grasped. The movement of the position of the light detection region is executed at. In addition, since it is easier to interpret the measurement result when the elapsed time during measurement and the movement distance of the position of the light detection region are in a proportional relationship, it is preferable that the movement speed is basically a constant speed. However, the present invention is not limited to this.

  By the way, with respect to the moving speed of the position of the light detection region, in order to quantitatively and accurately execute individual detection of luminescent particles from the measured time-series light intensity data, or counting of the number of luminescent particles. The moving speed is preferably set to a value faster than the random movement of the luminescent particles, that is, the moving speed due to the Brownian movement. Since the observation target particle of the optical analysis technique of the present invention is a particle that is dispersed or dissolved in a solution and moves freely and randomly, the position moves with time by Brownian motion. Therefore, when the moving speed of the position of the light detection region is slower than the movement due to the Brownian motion of the particle, the particle moves randomly within the region as schematically illustrated in FIG. As a result, the light intensity changes randomly as shown in FIG. 3B (as already mentioned, the excitation light intensity in the photodetection area decreases outward with the center of the area as the apex), and each individual. It becomes difficult to specify a significant change in light intensity corresponding to the luminescent particles. Therefore, preferably, as shown in FIG. 4 (A), the particle crosses the light detection region in a substantially straight line, so that the time-series light intensity data is illustrated in FIG. 4 (B). Thus, the light intensity change profile corresponding to each light emitting particle becomes substantially uniform (if the light emitting particle passes through the light detection region substantially linearly, the light intensity change profile is the excitation light intensity distribution). The movement speed of the position of the light detection region is the average movement speed (diffusion movement speed) due to the Brownian motion of the particles so that the correspondence between the individual luminescent particles and the light intensity can be easily identified. Set faster than.

Specifically, the time Δt required for the luminescent particles having the diffusion coefficient D to pass through the light detection region (confocal volume) having the radius Wo by Brownian motion is expressed by a relational expression (2Wo) ² = 6D · Δt (4)
From
Δt = ( ² Wo) ² / 6D (5)
Therefore, the speed (diffusion movement speed) Vdif at which the luminous particles move by Brownian motion is approximately
Vdif = 2Wo / Δt = 3D / Wo (6)
It becomes. Therefore, the moving speed of the position of the light detection region may be set to a value sufficiently faster than that with reference to the Vdif. For example, when the diffusion coefficient of the observation target particle is expected to be about D = 2.0 × 10 ⁻¹⁰ m ² / s, if Wo is about 0.62 μm, Vdif is 1.0 × Since 10 ⁻³ m / s, the moving speed of the position of the light detection region may be set to 15 mm / s, which is 10 times or more. In addition, when the diffusion coefficient of the observation target particle is unknown, the profile of the change in the light intensity by setting the moving speed of the position of the light detection region in various ways is expected (typically, the excitation light intensity distribution). A preliminary experiment for finding a condition that is substantially the same as that described above may be repeatedly performed to determine a suitable moving speed of the position of the light detection region.

(2) Analysis of light intensity When the time-series light intensity data of the sample solution is obtained by the above processing, the computer 18 stores the program stored in the storage device (the predetermined width in the time-series light intensity data). The procedure for calculating the characteristic value of the light intensity representing the presence or absence of light from a single light emitting particle for each time interval of, and the light signal from each light emitting particle based on the light intensity characteristic value for each time interval of a predetermined width The light intensity analysis as described below may be executed by the processing according to the procedure for individually detecting the light intensity.

(I) Detection of one luminescent particle In the time-series light intensity data, the locus when one luminescent particle passes through the light detection region is substantially linear as shown in FIG. In some cases, the change in the light intensity corresponding to the luminescent particle is a profile that reflects the light intensity distribution in the light detection region (determined by the optical system), as schematically depicted in FIG. Usually, it has a substantially bell-like shape, and is significantly different from data (a state in which noise is randomly generated) during a period in which no luminescent particles pass. Therefore, as already mentioned, in the present embodiment, the light intensity characteristic value is calculated for each time interval having a predetermined width on the time-series light intensity data. As the characteristic value of the light intensity, when the time-series light intensity data is time-series photon count data, as described above, the total value of the number of photons in the time interval of the predetermined width, the median value of the number of photons Particles calculated from the average value of photons, the standard deviation value of the number of photons, the dispersion value of the number of photons, the entropy value of the number of photons, the maximum value of the number of photons, and the autocorrelation function value of the number of photons. It may be a value selected from a group of numbers.

  More specifically, for each characteristic value, the total number of photons is the total number of photons in a predetermined time interval. Since the total value of the time interval in which the light signal of the luminescent particles exists is increased by the number of photons from the luminescent particles than the total value of the other time intervals, the total value of a certain time interval is larger than a predetermined threshold value. Is larger, it is determined that the light signal of the luminescent particles is present in the time interval.

  The median value and the maximum value of the number of photons are respectively the median value and the maximum value of the number of photons found in a time interval of a predetermined width. Since the number of photons is larger during the period when the light of the luminescent particles arrives at the photodetector than when only normal noise occurs, the median and maximum value of the time interval in which the light signal of the luminescent particles is present also increases. To do. Therefore, when the median value or the maximum value of a certain time interval is larger than a predetermined threshold, it is determined that the light signal of the luminescent particles exists in that time interval.

  The average value of the number of photons is a time average value of the number of photons in a time interval of a predetermined width. As described above, the total value of the time interval in which the light signal of the luminescent particles is present is increased by the number of photons from the luminescent particles than the total value of the other time intervals, and thus the number of photons in the time interval of a predetermined width. The time average also increases. Therefore, when the average value in a certain time interval is larger than the predetermined threshold, it is determined that the light signal of the luminescent particles exists in that time interval.

The standard deviation value, dispersion value, and entropy value of the number of photons are the standard deviation value, dispersion value, and entropy value of the information amount in the time average of the number of photons in the time interval of the predetermined width, respectively. This is a characteristic value representing the degree of variation in the time change of the number of photons found in the time interval. When the light signal of the luminescent particles is present in a certain time interval, the time change in the number of photons becomes more intense than in other time intervals. Accordingly, the standard deviation value, dispersion value, and entropy value of the number of photons in the time interval in which the light signal of the luminescent particles is present increase compared to the values in the other time intervals. When the value is larger than the predetermined threshold, it is determined that the light signal of the luminescent particles is present in the time interval. It should be noted that the entropy value of the number of photons is the number of times when the number of photons is x in a certain time interval when the probability that the number of photons at a certain time (BIN TIME) is x is px. ix
-Log ₂ (p0 ⁱ⁰ · p1 ^{i1 ····} px ^{ix ····} pn ⁱⁿ ) (7)
Is the value given by Usually, the probability px that the number of photons is x is p0>p1>p2>...> Px. The entropy value in the section where only noise exists is about -log ₂ (p0 ⁱ⁰ · p1 ⁱ¹ · p2 ⁱ² ), but in the section where the light signal of the luminescent particles exists, -log ₂ (p0 ⁱ⁰ · p1) ⁱ¹ , p2 ⁱ² , p3 ⁱ³ , p4 ⁱ⁴ ), etc., and the value increases.

により与えられる（ここで、τＤは、並進拡散時間であり、ＡＲは、ストラクチャ・パラメータであり、Ｎは、粒子数である。）。上記の式に於いて、相関時間τ＝０のときのＣ（０）から、粒子数Ｎは、１／（Ｃ（０）−１）により与えられる。この粒子数は、時間区間毎に見出される粒子数であると考えられるので、上記の特性値と同様に、所定の閾値よりも大きいとき、その時間区間に於いて発光粒子の光信号が存在すると判定される。The number of particles calculated from the autocorrelation function value of the number of photons with zero correlation time in a time interval of a predetermined width is an amount corresponding to the number of particles in that time interval. According to FCS theory, the autocorrelation function C (τ) of light intensity is

(Where τD is the translational diffusion time, AR is the structure parameter, and N is the number of particles). In the above formula, from C (0) when the correlation time τ = 0, the number N of particles is given by 1 / (C (0) −1). Since this number of particles is considered to be the number of particles found for each time interval, and similar to the above characteristic value, when the light signal of the luminescent particles is present in that time interval when it is larger than a predetermined threshold value. Determined.

(Ii) Counting of luminescent particles In the luminescent particle detection process described above, the predetermined width of the time interval is substantially larger than the time width required from the time when one luminescent particle enters the light detection region to the time when it escapes. One light emission when it is set to be longer and substantially shorter than the time width from the time when one light emitting particle enters the light detection region to the time when another light emitting particle enters the light detection region. Since the particles correspond to one time interval in which it is determined that the light signal of the luminescent particles is present, the number of luminescent particles is determined by counting the number of such time intervals.

(Iii) Determination of Number Density or Concentration of Luminescent Particles When counting of luminescent particles is performed, the number density or concentration of luminescent particles is determined using the total volume of the region through which the light detection region has passed. However, since the effective volume of the light detection region varies depending on the wavelength of the excitation light or detection light, the numerical aperture of the lens, and the adjustment state of the optical system, it is difficult to calculate from the design value. Therefore, in the present embodiment, the measurement of the light intensity and the luminescent particles described above are performed under the same conditions as the measurement of the sample solution to be inspected for the solution having a known concentration of the luminescent particles (control solution). Detection and counting, and from the number of detected luminescent particles and the concentration of the luminescent particles in the control solution, the total volume of the region through which the light detection region has passed, that is, the relationship between the number of detected luminescent particles and the concentration is determined You may be supposed to be. The luminescent particles of the control solution may preferably be luminescent labels (fluorescent dyes or the like) having the same wavelength characteristics as the observation target particles. Specifically, for example, for a control solution having a concentration C of luminescent particles, if the number of detected luminescent particles in the control solution is N, the total volume Vt of the region through which the photodetection region has passed is
Vt = N / C (9)
Given by. In addition, as a control solution, a plurality of solutions having different concentrations of luminescent particles are prepared, measurement is performed for each, and the calculated average value of Vt is adopted as the total volume Vt of the region through which the light detection region has passed. It may be like this. When Vt is given, the number density c of the luminescent particles of the sample solution whose counting result of the luminescent particles is n is
c = n / Vt (10)
Given by. The volume of the light detection region and the total volume of the region through which the light detection region has passed are given by any method, for example, using FCS or FIDA, without depending on the above method. It's okay. Further, in the optical analyzer of the present embodiment, the relationship between the concentration C of various standard luminescent particles and the number N of luminescent particles (formula (9)) regarding the assumed movement pattern of the light detection region. ) May be stored in advance in the storage device of the computer 18 so that the user can use the information stored as appropriate when the user of the device performs optical analysis.

  Thus, according to the above-described photoanalysis technique of the present invention, the position of the micro area which is the photo detection area in the sample solution is moved, that is, the sample solution is scanned while traversing the photo detection area. It does not include statistical processing such as calculation of fluctuations in fluorescence intensity performed in FCS, FIDA, etc. by the method of detecting luminescent particles individually or counting such luminescent particles. It is possible to detect the state or characteristics of the observation target particles in the sample solution whose particle concentration or number density is lower than the level handled by FCS, FIDA, or the like.

  Since the optical analysis technique of the present invention basically uses the same optical system as FCS, FIDA, etc., it may be executed in combination with FCS, FIDA, etc. For example, in the case of detecting an interaction between a plurality of types of substances in a solution containing a plurality of types of substances, and when the concentration difference between the substances is large, for example, the concentration of one of the substances is on the order of nM. Yes, when the other substance is on the order of pM, the high concentration substance is measured and analyzed by FCS or FIDA, and the low concentration substance is measured using the optical analysis technique of the present invention. And an analysis aspect such as performing an analysis will be performed. In such a case, it is advantageous to prepare a plurality of photodetectors as illustrated in FIG.

  In order to verify the effectiveness of the present invention described above, the following experiment was conducted. It should be understood that the following examples illustrate the effectiveness of the present invention and do not limit the scope of the present invention.

  As a luminescent particle, ATTO633 (Signa-Aldrich Cat. No. 18620) which is a fluorescent dye was used, and the measurable range of the concentration of the luminescent particle in the sample solution according to the present invention was verified. As a sample solution, a phosphate buffer (containing 0.05% Tween 20) was prepared for ATTO633 so that the concentrations thereof were 10 fM, 100 fM, 1 pM, 10 pM, and 100 pM.

  In the measurement by the optical analysis technique of the present invention, a single molecule fluorescence measurement device MF20 (Olympus Corporation) equipped with a confocal fluorescence microscope optical system and a photon counting system is used as the optical analysis device. In accordance with the embodiment described in “1) Measurement of Light Intensity of Sample Solution”, time-series light intensity data was obtained for each sample solution. As the objective lens, a water immersion objective lens (40 ×, NA = 1.15, WD = 0.26) was used. Note that the photodetector 16 uses an ultrasensitive photodetector that can detect the arrival of one photon, and light detection is performed sequentially for a predetermined unit time (BIN TIME) over a predetermined time. The photon counting is performed in such a manner that the number of photons arriving at the photodetector is measured. Therefore, the time-series light intensity data is time-series photon count data. The excitation light was 633 nm laser light, and the detection light wavelength was 660-710 nm using a bandpass filter. The moving speed of the position of the light detection region in the sample solution was 15 mm / second, BIN TIME was 10 μsec, and measurement for 2 seconds was performed three times.

  FIG. 5A shows an example of the entire time-series photon count data obtained by measurement for 2 seconds, and FIG. 5B shows one of the time-series photon count data obtained by measurement for 2 seconds. The part is shown enlarged. After the measurement of the light intensity, the detection of the light signal of the luminescent particles in the time series photon count data acquired for each sample solution is performed as shown in FIG. 5B. The entire area of the data is divided into time intervals having a width of 200 μs, and in each time interval, the total value of the number of photons and the number of photons are obtained as characteristic values of light intensity indicating the presence or absence of light from a single light emitting particle. Photon number, photon number standard deviation value (SD value), photon number dispersion value, photon number entropy value, photon number maximum value, and photon number autocorrelation with zero correlation time The number of particles calculated from the function value was calculated. As a result, for example, as shown in FIG. 5B, in the section (Window # 30, Window # 32) in which it is possible to visually determine that the light signal of the luminescent particles exists, the example characteristic values are The value was larger than the characteristic value of the section (Window # 31) in which it can be determined that the light signal of the luminescent particles does not exist.

Thus, after calculating the characteristic value of the light intensity for each time interval in the entire time-series photon count data, each of the above characteristic values exceeds the corresponding predetermined threshold value for one luminescent particle. Under the assumption that an optical signal is present, the number of time intervals exceeding a predetermined threshold was counted for each characteristic value. FIG. 6 shows the number of time intervals in which each characteristic value exceeds a predetermined threshold value for each solution. The threshold value of each characteristic value was as follows.
Photon number total value 100 Photon number median value 4.5 Photon number standard deviation value 3.5 Photon number dispersion value 12.5 Photon number entropy 105
Maximum number of photons 13 particles Number of particles (calculated from autocorrelation function value) 0.4 For comparison, the time-series photon count data includes the number of consecutive time regions in which the number of photons exceeds 2. (The number of binarized areas).

  Referring to FIG. 6, all of the example characteristic values increased as the concentration of the luminescent particles in the sample solution increased. However, with respect to the number of particles calculated from the autocorrelation function value of the number of photons with a correlation time of 0, no significant change was observed when the concentration of the luminescent particles was less than 1 pM. On the other hand, with regard to the number of binarized regions, no significant concentration dependency was obtained in the sample solution used for the measurement. Thus, according to the present invention, the above results indicate that the light intensity representing the presence or absence of light from a single light emitting particle for each time interval of a predetermined width in time-series light intensity data obtained by measuring light according to the present invention. It is suggested that the light from the light-emitting particles can be detected individually and the concentration can be determined by calculating the characteristic value of and referring to the characteristic value.

  Furthermore, in the above time-series photon count data, the influence of the number of detected luminescent particles by the width of the time interval for calculating the characteristic value was verified. FIG. 7 shows the number of detected luminescent particles (characteristic value) when the width of the time interval for calculating the characteristic value (total number of photons) is changed in the time-series photon count data of the sample solution having a luminescent particle concentration of 10 pM. Indicates the number of time intervals exceeding a predetermined threshold). Referring to the figure, when the time interval width was 100 to 200 μs, the number of detected luminescent particles was stable. On the other hand, when the time interval width was less than 100 μsec, the number of detected particles increased apparently. Under the measurement conditions of this experimental example, the time required for a single luminescent particle to linearly pass through the light detection region is calculated to be about 74 μsec. It is considered that the number of times that one luminescent particle is detected over two or more time intervals is reduced by making it longer than the time required for linearly passing through the detection region. In the above results, when the time interval width exceeded 200 μsec, the number of detected particles was apparently reduced. This is considered to be because when the time interval width is increased, the number of time intervals in which optical signals of a plurality of luminescent particles are present increases. Actually, in the above solution conditions and apparatus conditions, the upper limit ωhigh of the predetermined width of the time interval calculated with the allowable error of about 5% using the equation (2d) was about 200 μsec (light detection region) The radius Wo was set to 1 μm.) These results show that the width of the time interval is substantially longer than the time width required from the time when one luminescent particle enters the light detection region to the time when it exits, and substantially, This suggests that one luminescent particle can correspond to one time interval by setting it shorter than the time width from the time of entering the light detection region to the time of entering another light emitting particle into the light detection region. ing.

  Thus, according to the optical analysis technique of the present invention, it is possible to determine the number density or concentration of the luminescent particles up to a concentration range lower than the number density or concentration limit that can be measured by the method using the conventional fluorescence intensity. It was shown that. In addition, the lower limit of the particle concentration that can be measured by optical analysis techniques such as FCS, FIDA, and PCH including statistical processing such as calculation of fluctuations in fluorescence intensity was about 1 nM, whereas the measurement of this example. The lower limit of the possible particle concentration is -10 fM. Therefore, according to the present invention, it is shown that it is possible to measure particles in a concentration range that is significantly lower than in the case of optical analysis techniques such as FCS, FIDA, and PCH. It was done.

Claims (18)

An optical analyzer that detects light from luminescent particles that are dispersed in a sample solution and move randomly using an optical system of a confocal microscope or a multiphoton microscope,
A light detection region moving unit that moves the position of the light detection region of the optical system in the sample solution;
A light detection unit for detecting light from the light detection region;
Generate time-series light intensity data of light from the light detection area detected by the light detection unit while moving the position of the light detection area in the sample solution, and the time-series light intensity A signal processor for individually detecting optical signals from each of the luminescent particles in the data;
The signal processing unit has a predetermined width including a plurality of unit times in the time-series light intensity data, and is substantially required from the time when one light emitting particle enters the light detection region to the time when it escapes. It is set to be longer than the time width, and is substantially shorter than the time width from the time when one light emitting particle enters the light detection region to the time when another light emitting particle enters the light detection region. The number of particles calculated from the autocorrelation function value of the light intensity with the standard deviation of the light intensity, the dispersion of the light intensity, the entropy of the light intensity, and the correlation time as 0 for each time interval of the predetermined width set to be Calculating a characteristic value of light intensity representing the presence or absence of light from a single luminescent particle selected from the group, and individually detecting a light signal from each of the luminescent particles using the characteristic value Features device.   The apparatus according to claim 1, wherein the signal processing unit counts the number of time intervals of the predetermined width having the optical signal from the luminescent particles, and is detected while the position of the light detection region is moving. An apparatus for counting the number of the luminous particles.   3. The apparatus according to claim 1, wherein the light detection unit detects light from the light detection region by photon counting, and the time-series light intensity data is time-series photon count data. Device to do.   4. The apparatus according to claim 1, wherein the light detection area moving unit moves the position of the light detection area at a speed faster than a diffusion movement speed of the luminescent particles.   5. The device according to claim 1, wherein a characteristic value of the light intensity in the time interval of the predetermined width in which light from the single luminescent particle exists is the single luminescent particle. When the light intensity characteristic value is larger than a predetermined threshold value and greater than a predetermined threshold value, the light emission is performed in the predetermined width time period. An apparatus characterized in that an optical signal from a particle is determined to be present.   6. The apparatus according to claim 1, wherein the signal processing unit determines the number density or concentration of the luminescent particles in the sample solution based on the number of the detected luminescent particles. Device to do. An optical analysis method for detecting light from luminous particles dispersed and moving randomly in a sample solution using an optical system of a confocal microscope or a multiphoton microscope,
Moving the position of the light detection region of the optical system in the sample solution;
Detecting light from the light detection region while moving the position of the light detection region in the sample solution to generate time-series light intensity data;
In the time-series light intensity data, calculating a light intensity characteristic value indicating the presence or absence of light from a single luminescent particle for each predetermined time interval including a plurality of unit times ;
Individually detecting light signals from individual luminescent particles based on a characteristic value of light intensity for each time interval of the predetermined width,
The predetermined width is set to be substantially longer than a time width required from the time when one light emitting particle enters the light detection region to the time when it exits, and substantially, the light emitting particle of the one light emitting particle It is set to be shorter than the time width from the time of entering the light detection region to the time of entering another light emitting particle into the light detection region,
The characteristic value of the light intensity is calculated from the autocorrelation function value of the light intensity when the standard deviation of the light intensity, the dispersion of the light intensity, the entropy of the light intensity, and the correlation time are 0 in the time interval of the predetermined width. A method characterized in that the value is selected from a group of particles . 8. The method of claim 7 , further comprising: counting the number of time intervals of the predetermined width having a light signal from the luminescent particles, and detecting the number of the luminescent particles detected during movement of the position of the light detection region. A method comprising the step of counting. The method according to claim 7 or 8 , wherein light from the light detection region is detected by photon counting in the process of detecting light from the light detection region and generating time-series light intensity data. The time-series light intensity data is time-series photon count data. The method of any of claims 7 to 9, in the step of moving the position of the light detection region, the position of the light detection region is moved at a speed faster than the diffusion movement speed of the phosphor particles A method characterized by that. The method of any of claims 7 to 10, wherein the single characteristic value of at the light intensity to the time interval of the predetermined width light is present from luminescent particles said single light emitting particles When the light intensity characteristic value is larger than a predetermined threshold value and greater than a predetermined threshold value, the light emission is performed in the predetermined width time period. A method characterized in that it is determined that an optical signal from a particle is present. The method of any of claims 7 to 11, further based on the number of the detected luminescence particles, and characterized in that it comprises the step of determining the number density or concentration of the luminescent particles in the sample solution how to. A computer program for optical analysis for detecting light from luminous particles dispersed and moving randomly in a sample solution using an optical system of a confocal microscope or a multiphoton microscope,
A procedure for moving a position of a light detection region of the optical system in the sample solution;
A procedure for detecting light from the light detection region during movement of the position of the light detection region in the sample solution and generating time-series light intensity data;
In the time-series light intensity data, a procedure for calculating a characteristic value of light intensity representing the presence or absence of light from a single luminescent particle for each time interval of a predetermined width;
Causing the computer to execute a procedure for individually detecting light signals from individual light-emitting particles based on the characteristic value of light intensity for each time interval of the predetermined width,
The predetermined width is set to be substantially longer than a time width required from the time when one light emitting particle enters the light detection region to the time when it exits, and substantially, the light emitting particle of the one light emitting particle It is set to be shorter than the time width from the time of entering the light detection region to the time of entering another light emitting particle into the light detection region,
The characteristic value of the light intensity is calculated from the autocorrelation function value of the light intensity when the standard deviation of the light intensity, the dispersion of the light intensity, the entropy of the light intensity, and the correlation time are 0 in the time interval of the predetermined width. A computer program characterized by being a value selected from a group of particle numbers . 14. The computer program according to claim 13 , further comprising counting the number of time intervals of the predetermined width having a light signal from the light emitting particles, and detecting the light emitting particles detected during movement of the position of the light detection region. The computer program characterized by including the procedure which counts the number of. 15. The computer program according to claim 13 or 14 , wherein light from the light detection region is detected by photon counting in a procedure for detecting light from the light detection region and generating time-series light intensity data. The computer program characterized in that the time-series light intensity data is time-series photon count data. 16. The computer program according to claim 13 , wherein the position of the light detection region is faster than the diffusion movement speed of the light emitting particles in the procedure of moving the position of the light detection region of the optical system. A computer program which is moved by Be any computer program of claim 13 to 16, light emission characteristic value of at the light intensity to the time interval of the predetermined width light is present from the single light emitting particles of said single When the light intensity characteristic value is larger than a predetermined threshold value when the light intensity characteristic value is larger than a predetermined threshold value, the light intensity characteristic value is larger than a predetermined threshold value. A computer program characterized in that it is determined that an optical signal from a luminescent particle is present. It is any computer program of claim 13 or 17, further based on the number of the detected luminescence particles, comprising the steps of determining the number density or concentration of the luminescent particles in the sample solution A computer program.

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